Use of uridine triphosphates and related compounds for the prevention and treatment of pneumonia in immobilized patients

ABSTRACT

A method of promoting drainage of mucous secretions in the congested airways of a bedridden/immobilized patient or an intubated/mechanically ventilated patient is disclosed. The method comprises administering to the airways of the patient a uridine phosphate such as uridine 5′triphosphate (UTP) or P1,P4di(uridine5′) tetraphosphate, an analog of UTP, or any other analog, in an amount effective to promote drainage of fluid in the congested airways, including sinuses, to increase the ciliary beat frequency of cilia on the surface lumina epithelia cells, to increase the secretions of mucous by globlet cells and to promote the clearance of retained secretions by hydrating mucous secretions, by stimulating ciliary beat frequency in the airways and by stimulating surfactant production. Pharmaceutical formulations and methods of making the same are also disclosed. Methods of administering the same would include any liquid suspension (including nasal drops or eye drops or spray), oral form (liquid or pill), aerosol inhalation, powder form, topical, injected, intraoperatve instillation or suppository form.

TECHNICAL FIELD

[0001] This invention relates to a method of removing or preventing theaccumulation of retained mucous secretions from the lungs and bronchi ofimmobilized or bedridden patients, including those whose breathing isassisted by mechanical ventilation.

BACKGROUND OF THE INVENTION

[0002] Bedrest or immobility can result from a variety of healthproblems, both acute and chronic in nature. A primary concern in caringfor persons who are immobilized or placed on bedrest is that ofprevention of pneumonia and other respiratory problems. Once pneumoniadevelops in these patients, morbidity and mortality can be significant.Because of the immobility it may be difficult for patients to cough andmobilize secretions. Immobile patients include patients confined toeither beds or wheelchairs. In addition to complications arising fromthe immobility, the underlying health problem may place patients atincreased risk for infection. Factors or disease states which predisposefor high risk for pneumonia development include: altered conciousness(from head injury, anesthesia, drug overdose or other serious illness),tracheal intubation (via endotracheal, nasotracheal, or tracheostomytubes), mechanical ventilation, and other procedures or treatmentsincluding intra-aortic balloon pump, hemo- or ultrafiltration, chronicdisease states such as cancer, progressive neuromuscular disorders(multiple sclerosis, amytropic lateral sclerosis, etc.), heart disease,diabetes mellitus, acute neurological disorders (stroke, seizures,Guillain-Barre' syndrome, spinal cord injury), and rehabilitation frominjuries or surgeries (bedrest, traction, etc.). (p. 502“Medical-Surgical Nursing: Assessment and Management of ClinicalProblems” by S. Lewis and I. Collier, 2nd ed. 1987, McGraw-Hill, NewYork).

[0003] Mechanical ventilation is indicated for respiratory failure orcompromise resulting from a variety of pulmonary disorders andcomplications. It has been estimated that over 100,000 patients requiremechanical ventilation in the U.S. every year (I. Kappstein, et al.,Eur. J. Clin. Microbiol. Infect. Dis. 11(6), 504-8 (1992)). Morbidityand mortality from the underlying disorders can be high, and theaddition of mechanical ventilation further increases risk. Complicationsresulting from mechanical ventilation may include: ventilator-associatedpneumonia (VAP), pneumothorax, pulmonary embolus, right mainstembronchus intubation, accidental extubation, aspiration of gastriccontents, sepsis, fluid overload/heart failure, hypotension, and death(B. deBoisblanc, et al., Chest 103, 1543-7 (1993)). One of the mostcommon complications is VAP, with an incidence conservatively estimatedat 25%, with greater than 12,000 deaths per year due to VAP (D. Craven,et al., Am. Rev. Respir. Dis. 133, 792-6 (1986). Increased vigilance bynursing or other health care professionals, invasive monitoring, use ofvasoactive medications, and frequent overall assessments greatlyincrease the cost of care for mechanically ventilated patients. Aconservative estimate for total cost of these mechanically ventilatedpatients approaches $1.5 billion per year in the U.S. alone (I.Kappstein, supra).

[0004] Patients who are intubated and on mechanical ventilation are atseveral-fold higher risk for developing pneumonia and other pulmonarycomplications than non-intubated patients, due to the impairment orabsence of several aspects of the normal pulmonary defense mechanisms(T. Inglis, J. Hosp. Infect. 30, 409-13 (1995)). Normal defensemechanisms consist of: 1) filtration, warming, and humidification ofair; 2) epiglottis closure over the trachea; 3) cough reflex; 4)mucociliary escalator system; 5) immunoglobulins A and G; and 6)activity of alveolar macrophages. Airways distal to the larynx arenormally sterile, but with intubation, the cough reflex is impaired andclosure of the epiglottis cannot occur, allowing contamination of thelower airways. Because clinical practice guidelines generally do notadvocate the maintenance of a complete airway seal in the trachea by theendotracheal cuff, some leakage of nasopharyngeal secretions below theepiglottis may occur, therefore increasing risk for infection in thelower airways (P. Mahul, et al., Intensive Care Med. 18, 20-5 (1992)).

[0005] The leading cause of VAP is thought to be aspiration of colonizedgastric secretions via the incompletely closed glottis (P. Mahul, etal., supra). Colonization of the lower respiratory tract, especiallywith gram-negative bacteria is an early stage in the development of VAP.In addition, the use of suction catheters via the endotracheal tube toclear lower airway secretions, as well as other manipulations of theventilatory system, significantly increase the chance for nosocomialinfection, especially pneumonia. The normal warming, humidification, andfiltration mechanisms for distal airways are non-functional forintubated patients, and the underlying conditions of the patient, i.e.,malnutrition, fluid and/or electrolyte imbalance, and infections, mayfurther complicate a patient's prognosis.

[0006] Mucociliary transport velocity has been shown to be impaired inpatients who are intubated and receiving mechanical ventilation (F.Konrad, et al., Intensive Care Med. 21, 482-89 (1995); F. Konrad, etal., Chest 105(1), 237-41 (1994); F. Konrad, et al., Chest 102(5),1377-83 (1992)). Because movement and clearance of secretions is animportant lung defense mechanism, any impairment of this function, inaddition to the introduction of artificial airways, mechnicalventilation, and the underlying disease state, can severely compromisethe pulmonary host defense mechanisms.

[0007] Agents that can obviate the need for intubation and mechanicalventilation, or reduce time on mechanical ventilation, therebydecreasing the incidence of complications such as VAP, would certainlyhave a significant impact in the critical care setting, both in terms ofthe health of the patient and the costs associated with treatment.Applicants have discovered that uridine 5′-triphosphate (UTP) andrelated nucleotide compounds modulate specific activities of humanairway epithelial cells that are components of the mucociliaryescalator. Transport of foreign particles out of the lungs via themucociliary escalator relies on the integrated action of: 1) mucussecretion by goblet cells and submucosal glands which traps foreignparticles; 2) cilia to propel the mucus out of the lungs; and 3)epithelial ion transport systems which maintain the ionic milieu of, andhence the viscosity of, airway surface liquid to allow effective ciliarybeating. Application of extracellular UTP to the apical surface ofnormal human nasal epithelial cells in primary culture causes increasedCl— secretion in a concentration-dependent manner (S. Mason, et al., Br.J. Pharmacol. 103, 1649-56 (1991); M. Knowles, et al., N. Engl. J. Med.325, 533-8 (1991)). This response was also observed in cultured nasalepithelial cells from cystic fibrosis (CF) patients (R. Benali, et al.,Am. J. Respir. Cell Mol. Biol. 10, 363-8 (1994)). This increased Cl—transport has been associated with increased fluid transport across theepithelium (C. Jiang, et al., Science 262, 424-7 (1993)). In addition tothese effects on Cl— and fluid transport, UTP has been shown to producean increase in cilia beat frequency in cultured human epithelial cellsfrom normal adult humans and CF patients (D. Drutz, et al., Drug DevResearch 1996; 37(3):185 “Uridine 5′ Triphosphate (UTP) RegulatesMucociliary Clearance Via Purinergic Receptor Activation”, presented at“Purines '96” conference held in Milan, Italy, Jul. 6-9, 1996). Theseactions of UTP have been associated with an increase in intracellularcalcium ion (Ca++) due to stimulation of phospholipase C by the P₂Y₂receptor (H. Brown, et al., Mol. Pharmacol. 40, 648-55 (1991)). UTP hasalso been shown to increase the rate and total amount of mucin secretionby goblet cells in human airway epithelial explants (M. Lethem, et al.,Am. J. Respir. Cell Mol. Biol. 9, 315-22 (1993)). These effects wereobserved in tissues from both healthy individuals and patients with CF.

[0008] As for secondary pharmacological effects, aerosol administrationof UTP (10-2 M and 10-1 M in nebulizer) to anesthetized and ventilateddogs had no significant effects on peak inspiratory airway pressure,mean pulmonary artery pressure, heart rate, cardiac output, thoracicaortic pressure, electrocardiogram, or arterial blood gases (S. Mason,et al., Am. Rev. Respir. Dis. 147, A27 (1993)). To test the effect ofintravenous administration, sequential doses of intravenous UTP (0.1, 1,3 and 5 mmoles/kg) were infused into anesthetized, ventilated dogs over10 minutes produced no significant changes in mean pulmonary arterypressure, heart rate, cardiac output, or mean arterial pressure. Id.

[0009] Because UTP has been shown to acutely improve mucociliaryclearance (MCC) by 2.5-fold in normal volunteers without significanteffects (D. Drutz, supra), it is thought that MCC improvement inmechanically ventilated patients would prevent the pooling ofsecretions, the plugging of mucus, and the resulting infections andatelectasis. In addition, removal of pulmonary secretions by coughing orsuctioning may be enhanced by hydrating and thinning mucus secretions.UTP may, therefore, provide a safe adjunct or alternative tobeta-adrenergic agonists for enhancing the removal of lung secretions inmechanically ventilated patients. Additionally, the improvement in MCCwill enhance the patient's pulmonary host defense mechanisms, thuspreventing ventilator-associated pneumonia (VAP) and other pulmonarycomplications, such as atelectasis. In addition, by acting on receptorsin Type II alveolar cells, UTP may enhance surfactant production andtherefore help maintain optimal gas exchange and airway epitheliumfunction in terminal small airways.

[0010] Applicant postulates that MCC in mechanically-ventilated patientscan be improved by administering UTP and its related compounds as wellas other nucleoside phosphates such as:P1,P4-di(uridine-5′)tetraphosphate (U2P4); adenosine 5′-triphosphate(ATP); 1,N6-ethenoadenosine 5′-triphosphate; adenosine 1-oxide5′-triphosphate; 3,N4-ethenocytidine 5′-triphosphate; orP1,P4-di(adenosine-5′)tetraphosphate (A₂P₄) to the site of fluidcongestion. UTP and U2P4 are the preferred embodiments of the presentinvention. By administering UTP or U2P4 prior to or soon afterintubation, VAP and other associated complications of mechanicalventilation may be avoided. The method of the present invention may alsobe used to treat chronic bronchitis patients who develop respiratorydistress that requires intubation. Finally, the method of the presentinvention may also be used to promote the drainage of retained mucoussecretions in immobilized or bedridden patients.

SUMMARY OF THE INVENTION

[0011] A method of preventing or treating pneumonia, includingventilator-associated pneumonia (VAP), in a subject in need of suchtreatment is disclosed. The method of the present invention may also beused to promote the drainage and clearance of retained mucous secretionsin immobilized or bedridden patients to prevent pneumonia. The methodcomprises administering to the patient a compound of Formula I, or apharmaceutically acceptable salt thereof, in an amount effective tohydrate mucous secretions and stimulate ciliary beat frequency in theluminal epithelial cells of the airway passages:

[0012] wherein:

[0013] X₁, X₂, and X₃ are each independently either O— or S—.Preferably, X₂ and X₃ are O—.

[0014] R₁ is O, imido, methylene, or dihalomethylene (e.g.,dichloromethylene, diflouromethylene). Preferably, R₁ is oxygen ordifluoromethylene.

[0015] R₂ is H or Br. Preferably, R₂ is H. Particularly preferredcompounds of Formula I are uridine 5′-triphosphate [UT] and uridine5′-O-(3-thiotriphosphate) [UTPγS].

[0016] In addition to Formula I, Formula II, i.e.,P1,P4di(uridine-5′)tetraphosphate [U2P4] is also a preferred embodimentof the invention. Another compound of Formula II isP1,P4-di(adenosine-5′)tetraphosphate [A₂,P₄]. The method of the presentinvention can also include administering a compound of Formula III(adenosine 5′ triphosphate [ATP] or 1,N6-ethenoadenosine 5′-triphosphateor adenosine 1-oxide 5′-triphosphate), or Formula IV(3,N4-ethenocytidine 5′-triphosphate).

[0017] wherein:

[0018] B is uracil or adenine, attached as in Formulae I and III.

[0019] wherein:

[0020] R₁, X₁, X₂, and X₃ are defined as in Formula I.

[0021] R₃ and R₄ are H while R₂ is nothing and there is a double bondbetween N-1 and C-6 (adenine), or

[0022] R₃ and R₄ are H while R₂ is O and there is a double bond betweenN-1 and C-6 (adenine 1-oxide), or

[0023] R₃, R₄ and R₂ taken together are —CH═CH—, forming a ring from N-6to N-1 with a double bond between N-6 and C-6(1,N6-ethenoadenine).

[0024] wherein:

[0025] R₁, X₁, X₂, and X₃ are defined as in Formula I.

[0026] R₅ and R₆ are H while R₇ is nothing and there is a double bondbetween N-3 and C-4 (cytosine), or,

[0027] R₅, R₆ and R₇ taken together are —CH═CH—, forming a ring from N-3to N-4 with a double bond between N-4 and C-4(3,N4-ethenocytosine).

[0028] A second aspect of the present invention is a pharmaceuticalformulation containing the compound of Formula I, II, III or IV in anamount effective to promote or enhance clearance of secretions, hydratemucous secretions and stimulate ciliary beat frequency in the luminalepithelial cells of the airway passages in a patient in need of suchtreatment.

[0029] A third aspect of the present invention is the stimulation ofsurfactant production in Type II alveolar cells.

[0030] A fourth aspect of the present invention is the use of the activecompounds disclosed herein for the manufacture of a medicament for thetherapeutic hydration of mucous secretions and stimulation of ciliarybeat frequency in the luminal epithelial cells of the airway passages ina patient in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 represents the results of the studies described in Example2 showing the effects of UTP on trachael mucous velocity.

[0032]FIG. 2 represents the results of the studies described in Example2 showing the effects of U₂P₄ on tracheal mucous velocity.

[0033]FIG. 3 represents the results of studies described in Example 2showing a bar graph of the TMV post dose for varying concentrations ofUTP and U₂P₄.

[0034]FIG. 4 represents the results of the studies described in Example3 showing the effects of UTP and U₂P₄ in mucociliary clearance of adultewes

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0035] The method of the present invention may be used to prevent ortreat pneumonia, including ventilator-associated pneumonia (VAP), byhydrating retained mucous secretions, stimulating ciliary beat frequencyand promoting clearing of mucous in the airways of a subject in need ofsuch treatment. The method of the present invention may also be used toprevent or treat sinusitis in nasally intubated patients, and to improvemucociliary clearance (MCC) thereby preventing pneumonia in chronicallyimmobilized or bedridden patients. The present invention increasesmucociliary clearance (MCC) in three ways: (1) by increasing the ciliarybeat frequency of cilia on the surface of luminal epithelia cells, (2)by increasing the secretions of mucins by goblet cells, and (3) byincreasing the chloride ion secretion and simultaneously increasing thesecretion of water into the periciliary liquid layer by luminalepithelial cells, consequently lowering the viscosity of the mucus. Themucins secreted by goblet cells form a layer on top of the cilia andcapture foreign particles, including viruses and bacteria; the mucinlayer is transported by the wave-like action of cilia; and the movementof cilia is facilitated by the hydration of the periciliary liquid layersurrounding the cilia. Further, the method of the present invention maybe used with the compounds disclosed to influence agonist activity onP₂Y₂ receptors thereby stimulating surfactant production.

[0036] Compounds illustrative of the compounds of Formula I aboveinclude: (a) uridine 5′-triphosphate (UTP); (b) uridine5′-O-(3-thiotriphosphate) (UTPγS); and (c) 5-bromo-uridine5′-triphosphate (5-BrUTP). These compounds are known or may be made inaccordance with known procedures, or variations thereof which will beapparent to those skilled in the art. For example, UTP may be made inthe manner described in Kenner, et al., J. Chem. Soc. 1954, 2288.Following this well-known methodology, UTP can be synthesized bycondensing 2′:3′-di-O-acetyl- or 2′:3′-O-isopropylidine-uridine-5′benzyl phosphorochloridate with a salt of tribenzyl pyrophosphate andthen removing the benzyl and other protecting groups. UTP has also beensynthesized by treating at room temperature a mixture of UMP and 85%orthophosphoric acid with excess DCC in aqueous pyridine. Hall andKhorana, J. Am. Chem. Soc. 76, 5056 (1954).

[0037] The Merck Index, Monograph No. 9795 (11th Ed. 1989) gives thechemical structure of UTP:

[0038] S-2-carbamoylethyl thiophosphate can be used for the chemicalsynthesis of thiophosphate compounds such as UTPγS which has a sulfur atits terminal phosphorus atom. S. Goody and F. Eckstein, J. Am. Chem.Soc. 93, 6252 (1971).

[0039] For simplicity, Formulae I-IV herein illustrate the activecompounds in the naturally occuring D-configuration, but the presentinvention also encompasses compounds in the L-configuration, andmixtures of compounds in the D- and L-configurations, unless otherwisespecified. The naturally occuring D-configuration is preferred.

[0040] Compounds illustrative of the compounds of Formula II include(P1,P4-di(adenosine-5′)tetraphosphate (A₂P₄) orP1,P4-di(uridine-5′)tetraphosphate U₂P₄). These compounds can be made inaccordance with known procedures, or variations thereof. For example,A₂P₄ was synthetically prepared by activating ADP withcarbonyldiimidazole. E. Rapaport, et al., Proc. Natl. Acad. Sci. USA72(2), 838-42 (1981). Treating aqueous solutions of adenosine-5′-mono-,di-, or triphosphate with carbodiimide results indiadenosine-5′-5′-polyphosphate (including A₂P₄). K. Ng and L. E. Orgel,Nucleic Acids Res. 15 (8),3572-80 (1987). U₂P₄ can be synthesizedthrough the reaction of uridine 5′-phosphoromorpholidate (0.54 mmol)with triethylamine salt of pyrophosphate (0.35 mmol) in a medium ofanhydrous pyridine (10 ml). C. Vallejo, et al., Biochem. Biophys. Acta438, 304-09 (1976).

[0041] Compounds illustrative of the compounds of Formula III aboveinclude (a) adenosine 5′-triphosphate (ATP) and (b) 1,N6-ethenoadenosine5′-triphosphate. Compounds illustrative of the compounds of Formula IVabove include (a) cytidine 5′-triphosphate and (b) 3,N4-ethenocytidine5′-triphosphate. These compounds can be made in accordance with knownprocedures, or variations thereof which will be apparent to thoseskilled in the art. For example, nucleoside triphosphates can besynthesized by the reaction of the phosphorimidazolidate formed from anucleotide and 1,1′-carbonyldiimidazole with inorganic pyrophosphate. D.Hoard and D. Ott, J. Am. Chem. Soc. 87, 1785-1788 (1965). Anothergeneral method involves adding a 2′,3′-iso-propylidene nucleoside to acold mixture of trialkyl, phosphate and phosphoryl chloride withstirring. The mixture is converted into the corresponding5′-phosphorodichloridate. 5′nucleotide is obtained by rapid hydrolysisof the chloridate group followed by removal of the isopropylidene groupat 70° C. M. Yoshikawa, et al., Tetrahedron Lett. 5065-68 (1967) andidem., Bull. Chem. Soc. (Jpn) 42, 3505-08 (1969).

[0042] Nucleoside-5′ phosphoramidates may be used as an improved methodfor the preparation of nucleoside-5′ polyphosphates. J. Moffatt and H.Khorana, J. Am. Chem. Soc. 83, 649-59 (1961); and B. Fischer, et al., J.Med. Chem. 36, 3937-46 (1993).

[0043] Etheno derivatives of cytidine and adenosine are prepared byknown methods. For example, a reaction using chloroacetaldehyde and thenucleosides adenosine and cytidine is well known. Chloroacetaldehydereacts with 9-N-methyladenine and I-N-methylcytosine in weakly acidicaqueous solutions to form ethenoderivatives of cytidine and adenosine.N. Kotchetkov, et al., Tetrahedron Lett. 1993 (1971); J. Barrio, et al.,Biochem. Biophys. Res. Commun. 46, 597 (1972); J. Secrist, et al.,Biochemistry 11, 3499 (1972); J. Bierndt, et al., Nucleic Acids Res. 5,789 (1978); K. Koyasuga-Mikado, et al., Chem. Pharm. Bull. (Tokyo) 28,932 (1980).

[0044] Derivatives with alpha, beta and gamma thiophosphorus groups canbe derived by the following or by adapting the following methods:2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one can be used tophosphitylate the 5′-hydroxy group of a nucleoside to form anintermediate, which on a subsequent reaction with pyrophosphate forms,in a double displacement process a P²,P³-dioxo-P′-5′-nucleosidylcyclotriphophite. Oxidation with sulfur forms a nucleoside5′-(1-thiocyclotriphosphate), which is hydrolyzed to the diastereomericmixture of a nucleoside 5′-O-(1-thiotriphosphate). Alternatively,P²,P³-dioxo-P′-5′-nucleosidylcyclo triphophite can be oxidized withiodine/water to yield nucleoside 5′-triphosphates. This reagent can alsobe used for the synthesis of nucleoside 2′,3′-cyclic phosphorothioates.J. Ludwig and F. Eckstein, J. Org. Chem. 54, 631-35 (1989).

[0045] Derivatives with alpha, beta and gamma thiophosphorus groups canalso be made by following the protocol recited in F. Eckstein and R.Goody, Biochemistry 15, 1685 (1976). [³⁵S]Adenosine5′(0-1-thiotriphosphate). [³⁵S]Adenosine 5′-phosphorothioate (7500 A₂₆₀units, 0.5 mmol) was converted to the pyridinium salt by passage overMerck I ion exchanger (pyridinium form). The solution was evaporated todryness, tri-n-octylamine (0.22 ml, 0.5 mmol) and methanol (ca. 10 ml)were added, and the mixture was stirred until a clear solution wasobtained. After evaporation, the residue was evaporated (three times)with dry dimethylformamide using an oil pump. The residue was dissolvedin anhydrous dioxane (2 ml) and diphenyl phosphorochloridate (0.15 md,0.75 mmol) and tri-n-butylamine (0.25 ml, 1 mmol) were added. Afterstirring the mixture at room temperature for 3 h, the solvent wasremoved by evaporation, and anhydrous ether (10 ml) and petroleum ether(30 ml) were added to the residue, and the mixture was left at 0° C. for30 min. The ether was decanted, the remaining material dissolved inanhydrous dioxane (1 ml), and the solution evaporated.

[0046] Tetrasodium pyrophosphate decahydrate (2.23 g, 0.5 mmol) wasconverted to the pyridinium salt by addition of tri-n-butylamine (2.43ml, 10 mmol) and evaporation to dryness to the tri-n-butylammonium salt.After repeated evaporation with anhydrous pyridine (three times), thematerial was dissolved in anhydrous pyridine (3 ml) and added to theactivated [³⁵S]adenosine 5′-phosphorothioate described above.

[0047] After stirring at room temperature for 2 h, ether (10 ml) wasadded to precipitate the product. The precipitate was dissolved in waterand chromatographed on a DE-52 cellulose column (37×2.5 cm), with alinear gradient of 1.5 l. Each of 0.05 M and 0.5 M triethylammoniiumbicarbonate. The product was eluted at ca. 0.33 M buffer, yield 1550A₂₆₀ units (0.1 mmol, 20%). For further purification this material wasrechromatographed on a QAE-A 25 Sephadex column (1.5×25 cm) with alinear gradient of 800 ml each of 0.25 M and 0.5 M triethylammoniumbicarbonate: yield 1200 A₂₆₀ units (16%). The material was not degradedby snake venom phosphodiesterase but was degraded to AMPS¹ by alkalinephosphatase under conditions described for ATPβS:

[0048] [³⁵S]Adenosine 5′-(O-1-Thiodiphosphate). The synthesis of thiscompound was carried out as described for [³⁵S]adenosine5′-(O-1-thiotriphosphate) except that phosphate was added to theactivated [³⁵S]adenosine 5′-phosphorothioate instead of pyrophosphate:yield 1410 A₂₆₀ units (0.94 mmol, 18%).

[0049] Adenosine 5′-(O-2-thiotriphosphate) (ATPβS) Adenosine5′-(O-2-thiodiphosphate (1.5 mmol; Goody and Eckstein, 1971) wasconverted to its pyridinium salt by passage over Merck I ion exchanger(pyridinium form) in methanol-water (1:1, v/v), and the solution wasevaporated to dryness using a rotary evaporator. Tri-n-octylamine (1.3ml, ca. 3 mmol) and methanol (10 ml) were added to the residue, and themixture was stirred until solution was obtained (ca. 30 min). Afterremoval of solvent under reduced pressure, the residue was dissolved indry pyridine (10 ml) and evaporated to dryness on a rotary evaporatorusing an oil pump. This process was repeated three times.

[0050] β-Cyanoethyl phosphate (Ba²⁺ salt, 854 mg, 3 mmol) was convertedto its mon(tri-n-octylammonium) salt in a similar way to that describedabove, using 3 mmol of tri-n-octylamine. The salt was dried by repeatedaddition and reevaporation of dry dimethylformamide (10 ml) and thendissolved in dry dioxane (15 ml). Diphenyl phosphorochloridate (0.9 ml,4.5 mmol) and tri-n-butylamine (0.45 ml) were added, and the solutionwas allowed to stand at room temperature for 3 h. After removal ofdioxane under reduced pressure, ether (30 ml) followed by petroleumether (60 ml; 60-80° C.) was added and, after shaking for a few minutes,the mixture was allowed to stand for 15 min in ice. The ether was thenremoved by decantation and the residue dissolved in dry dioxane (5 ml)which was then removed by evaporation under reduced pressure, and to theresidue was added the ADPβS tri-n-octylammonium salt, prepared asdescribed above dissolved in a mixture of dry hexamethylphosphorotriamidate (4 ml) and dry pyridine (4 ml). The resultingsolution was allowed to stand for 3 h at room temperature, and pryidinewas then removed under reduced pressure. The remaining mixture wastreated with 0.5 N sodium hydroxide (100 ml) for 1 h at roomtemperature, after which time the solution was neutralized by additionof Merck I ion exchanger (pyridinium form). The neutralized solution wasapplied to a column of DE-52-cellulose (ca. 50×4 cm) which was elutedwith a linear gradient of triethylammonium bicarbonate (0.1-0.35 M, 2×2l.). The product was eluted at about 0.25 M: yield 2750 A₂₆₀ units(12%); for ³¹PNMR spectrum, see Table I. This was slightly contaminatedwith ADPβS. A further 800 A₂₆₀ units of product were obtained, moreheavily contaminated with ADPβS. Pure ADPβS could be obtained bychromatography on DEAE-Sephadex A-25, using a gradient oftriethylammonium bicarbonate (0.2-0.5 M) at 4° C. The product behavedidentically with regard to TLC on PEI-cellulose with ATPβS synthesizedusing pyruvaate kinase.

[0051] R. Goody and F. Eckstein, J. Am. Chem. Soc. 93, 6252 (1971)describe the synthesis of thiophosphate analogs of nucleoside di-andtriphosphates having a sulfur at the terminal phosphorus atom by the useof S-2-carbamoylethyl thiophosphate.

[0052] Compounds of Formulas I, III, or IV where R₁ is CCl₂ and CF₂ canbe prepared by methods similar to that described in G. Blackburn, etal., J. Chem. Soc. Perkin Trans. I, 1119-25 (1984).

[0053] Adenosine 5′-(β,γ-μ-Difluoromethylene)triphosphate, AMPPCF₂P(1c).—(a) Morpholine-4-N,N′-dicyclohexylcarboxamidiniumadenosine-5′-phosphoromorpholidate (390 mg, 0.5 mmol) was dissolved inanhydrous, amine-free pyridine (5 ml). The solution was evaporated todryness and the procedure repeated twice more with exclusion ofmoisture. It was finally dissolved in pyridine (3 ml). Similarly thebis(tri-n-butylammonium) salt of difluoromethylenebisphosphonic acid(580 mg, 1.0 mmol) was evaporated repeatedly from its solution inpyridine (3×5 ml). Finally, the two pyridine solutions were combined andevaporated to dryness. The residue was kept in anhydrous pyridine (4 ml)for 24 h with magnetic stirring and exclusion of moisture. After thistime the solution was evaporated to remove pyridine. The residue wasdissolved in deionized water (5 ml), applied to a column ofDEAE-Sephadex (3×30 cm), and the product eluted with a linear saltgradient (0-0.5M-LiCl). Fractions containing the analogue were combinedand evaporated to dryness. The white solid residue was dissolved in asmall volume of anhydrous methanol (5 ml) and the nucleotideprecipitated out by the addition of a acetone (25 ml). The precipitatedproduct was collected by centrifugation and the whole procedure repeatedfour times. The white pellet was finally redissolved in methanol (10 ml)and evaporated to dryness to yield the white, powdery product as thetetralithium salt (164 MG, 54%), m.p. 225-235° C. (decomp.).

[0054] Adenosine 5′-(β,γ-μ-Dichloromethylene)triphosphate,AMPPCCl₂P(1e). Adenosine-5′-phosphoromorpholidate (390 mg, 0.5 mmol) wascondensed with the bis(tri-n-butylammonium) salt ofdichloro-methylenebisphosphonic acid (613 mg, 1 mmol). The product waschromatographed on DEAE Sephadex using a linear gradient (LiCl, 0-0.5 M,pH 7.0, 2 1). Fractions containing the product were combined andevaporated to dryness and the product isolated by repeated dissolutionin methanol (5 ml) and precipitation with acetone (25 ml) to give awhite powder (24 mg, 75.7%), m.p. 235-245° C. (decomp).

[0055] Preparation of guanosine 5′-(β,γ-μ-Difluoromethylenetriphosphate)GMPPCF₂P (2b)—Morpholine-4-N,N′-dicyclohexyl-carboxamidiniumguanosine-5′-phosphoromorpholidate (390 mg, 0.5 mmol) was dissolved in amixture of anhydrous pyridine (5 ml) and freshly distilled2-chlorophenox (4 ml). To this was added the bis(tri-n-butylammonium)difluoromethylenebisphosphonate (580 mg, 1 mmol). The solution wasstirred for 4 days with exclusion of moisture and light. After this timewater (50 ml) was added and the solution extracted with ether (3×50 ml).The aqueous phase was evaporated to dryness and the gummy residueredissolved in water (5 ml), applied to a column of DEAE Sephadex, andeluted with a linear salt gradient (LiCl, 0-0.5 M, pH 7.0). Fractionscontaining the analogue were combined and evaporated to dryness and theproduct repeatedly precipitated from methanol with acetone. Evaporationto dryness of the final product in methanol yielded the title compoundas a white powder (136 mg, 42.8%), m.p. 245-255° C. (decomp.).

[0056] Guanosine 5′(β,γ-μ-Dichloromethylene)triphosphate GMPPCCl₂P(2d).—In a reaction exactly analogous to that for (2b),morpholine-4-N,N′-dicyclohexylcarboxamidiniumguanosine-5′-phosphoromorpholidate (144 mg, 0.2 mmol) was combined withthe bis(tri-n-butylammonium) salt of dichloro-methylenebisphosphonicacid¹⁰ (360 mg, 0.6 mmol) to yield the product as a white powder, (81mg, 62%), m.p. 240-260° C. (decomp).

[0057] Compounds of Formula I, II, III where R₁ is CH2 can be preparedby methods similar to that described in T. Myers, et al., J. Am. Chem.Soc. 85, 3292-95 (1963). This methodology demonstrates that thesyntehsis of 5′-adenylylmethylene or diphosphonate has been accomplishedby the reaction of adenosine 5′-phosphoromidate withmethylenediphosphonic acid and by the condensation of AMP withmethylenediphosphonic acid in the presence of excessdicyclohexylcarbodiimide.

[0058] In addition, UTP, ATP, CTP, A₂P₄, 3,N⁴-ethenocytidinetriphosphate, 1,N⁶-ethenoadenine 5′-triphosphate, adenosine 1-oxide5′-triphosphate, ATPγS, ATPβS, ATPαS, AMPPCH₂P, AMPPNHP,N⁴-ethenocytidine and 1,N⁶-ethenoadenosine are commercially available,for example, from Sigma Chemical Company, PO Box 14508, St. Louis, Mo.63178.

[0059] The active compounds of Formulae I-IV may be administered bythemselves or in the form of their pharmaceutically acceptable salts,e.g., an alkali metal salt such as sodium or potassium, an alkalineearth salt, or an ammonium and tetraalkyl ammonium salts, NX₄+ (whereinX is C₁₋₄). Pharmaceutically acceptable salts are salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects.

[0060] The active compounds disclosed herein may be administered to thelungs, sinuses, ears or eyes by a variety of suitable means, but arepreferably administered by administering a liquid/liquid suspension(either a nasal spray of respirable particles which is either inhaled bythe subject or administered to the subject by means of nebulizationthrough the mechanical ventilation system, or nasal drops of a liquidformulation, or eye drops of a liquid formulation) comprised of theactive compound. Liquid pharmaceutical compositions of the activecompound for producing a nasal spray or nasal powder, nasal or eyedrops, or a liquid nebulized preparation may be prepared by combiningthe active compound with a suitable vehicle, such as sterile pyrogenfree water or sterile saline by techniques known to those skilled in theart. Further, other methods of administration could be used including,systemic administration and oral forms (liquid or pill), powderinhalation, topical, injectable, intra-operative instillation of a gel,cream, powder, foam, crystals or liquid suspension or suppository form.

[0061] The methods described herein are also applicable to veterinaryuse.

EXPERIMENTAL Example 1 Treatment of Patients at Risk ForVentilator-Associated Pneumonia (VAP)

[0062] Uridine 5′-triphosphate (UTP) or P1,P4di(uridine-5′)-tetraphosphate (U2P4) is administered to adult patientswith acute neurological impairment requiring intubation and mechanicalventilation. UTP is administered in an aerosolized form via an in-linenebulizer, 2-3 times per day, for a total of 5 days. The concentrationof UTP is in the range of 10-7 to 10-1 moles/liter. Treatment with UTPbegins within 12 hours of intubation/mechanical ventilation. The lengthof treatment for each patient is 5 days.

[0063] The safety of UTP to prevent or treat VAP is assessed by standardsafety measures of vital signs—heart rate, respiratory rate, bloodpressure, electrocardiogram and laboratory blood tests (e.g., bloodchemistries, complete blood count, hematology), as well as any adverseevents observed.

[0064] The effectiveness of UTP in preventing VAP is measured by adimunition of symptoms of VAP as determined by periodic physicalexaminations, and by laboratory and bacteriology evaluations. Anothermeans of measuring effectiveness is a decrease in the total number ofdays on mechanical ventilation—this is because an improvement inmucociliary clearance would decrease airway ventilating pressures andthe need for assisted ventilation.

Example 2 Trachael Mucus Study

[0065] The effects of UTP and U₂P₄ on trachael mucus velocity (TMV) werestudied using the following procedures: The nasal passages of consciousadult ewes were anesthetized with a 2% lidocaine solution, After localanesthesia was produced, a modified endotracheal tube 7.5 mm was placedsuch that the cuff was just below the vocal cords (verified byfluoroscopy). Inspired air was warmed and humidified. The cuff of theendotracheal tube was inflated only during administation of the testcompound to minimize possible impairment of TMV by the cuff. Testcompounds were administered by nebulization in a volume of 4 mL over aperiod of 10-12 min.

[0066] TMV was measured by fluroscopy. Ten to twenty radiopaque disks(Teflon®/bismuth trioxide; 1 mm diameter, 0.8 mm thick, weighing 1.8 mg)were introduced into the trachea through a modified suction catheterwith a puff of compressed air (3-4 L/min). Velocities of the individualdisks were recorded on videotape from a portable image intensifier unit.Individual disk velocities were calculated by measuring the distancetraveled by each disk during a 1 min observation period. Values reportedare the means of the individual disk velocities. A collar was worn bythe sheep which was used as a standard to correct for magnificationerrors inherent in the fluoroscope.

[0067] Both UTP and U₂P₄ produced significant dose-related effects ontracheal mucus velocity. The doses ranged from 4 to 400 μmole. Bothcompounds had their maximal effects at a dose of 400 μmole (4 ml of10⁻¹M). UTP produced a maximal effect of 125±7% of baseline(mean±standard error, n=6). U₂P₄ produced a maximal effect of 144±9% ofbaseline (n=6). Both compounds produced their maximal effects 15 minafter administration. The highest dose of UTP produced significanteffects on TMV up to 4 h after administration. The effects of U₂P₄ weresignificant out to 2 h after administration. Results are shown in FIGS.1-3.

Example 3 Mucociliary Clearance Study

[0068] In this study healthy adult ewes were given 99 mTc-labeled humanserum albumin (99 mTc-HSA) via a nebulized aerosol. The 99 mTC-HSA (20mCi) was administered over 5 min through a nasotracheal tube introducedunder local anesthesia with 2% lidocaine. After administration of the 99mTc-HSA, the animals were given a test compound: either UTP or U₂P₄.Test compounds were administered by nebulization in a volume of 4 mLover a period of 10-12 min. The test compounds were given at a dose of400 μmole. After the administration of the test compound, the animalswere extubated. Clearance of the radiolabeled particles was monitoredwith a gamma camera. Measurements were made at 0, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 76, 90, 105 and 120 min. Initial results (n=2)have shown that both test compounds promote clearance of theradiolabeled particles (compared to the saline control). Results areshown in FIG. 4.

[0069] The results of the studies in sheep on tracheal mucus velocity(TMV) and whole lung mucociliary clearance (WLC) demonstrated that UTPand U₂P₄ can enhance mucociliary clearance in intubated animals.Intubation is known to have detrimental effects on mucociliaryclearance. This was shown in the TMV study by the decline in TMV overthe study period in the saline treated animals. Despite this decliningbaseline, UTP and U₂P₄ were able to produce an enhancement of TMV.Although the intubation period was brief in the WLC study (only duringadministration of the test compound), impairment of mucociliaryclearance is a realistic possibility. UTP and U₂P₄ produced enhancedclearance under these conditions as well. These data strongly suggestthat these agents will enhance mucociliary clearance in intubatedpatients, which may be therapuetically useful in the prevention ortreatment of VAP and subjects at risk.

[0070] The subject methods and compounds decribed herein provide a meansfor preventing or treating ventilator-associated pneumonia in theintensive care unit setting. The method comprises administering to theairways of the subject a uridine triphosphate such as uridine5′-triphosphate (UTP) or any analog of UTP, for example U₂P₄, in anamount effective to hydrate mucous secretions to promote or enhanceclearance, or to stimulate ciliary beat frequency in the lungs.

[0071] The invention now being fully decribed, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. A method of preventing or treating pneumonia,including ventilator-associated pneumonia, in a bedridden or immobilizedsubject in need of such treatment, said method comprising: administeringto the subject a compound of Formula I, II, III or IV, or apharmaceutically acceptable salt thereof, in a pharmaceutical carrierhaving an amount of said compound effective to promote clearance fromthe airways:

 wherein: X₁, X₂, and X₃ are each independently selected from the groupconsisting of OH and SH; R₁ is selected from the group consisting of O,imido, methylene, and dihalomethylene; and R₂ is selected from the groupconsisting of H and Br;

 wherein: B is uracil or adenine, attached as in Formulae I and III;

 wherein: R₁, X₁, X₂, and X₃ are defined as in Formula I, R₃ and R₄ areH while R₂ is nothing and there is a double bond between N-1 and C-6(adenine), or R₃ and R₄ are H while R₂ is O and there is a double bondbetween N-1 and C-6 (adenine 1-oxide), or R₃, R₄, and R₂ taken togetherare —CH═CH—, forming a ring from N-6 to N-1 with a double bond betweenN-6 and C-6 (1,N6-ethenoadenine);

 wherein: R₁, X₁, X₂, and X₃ are defined as in Formula I, R5 and R6 areH while R7 is nothing and there is a double bond between N-3 and C-4(cytosine), or, R5, R6 and R7 taken together are —CH═CH—, forming a ringfrom N-3 to N-4 with a double bond between N-4 and C4(3,N4-ethenocytosine).
 2. A method according to claim 1, wherein saidcompound is delivered by administering a liquid/liquid suspension,including eye drops of said compound to the eyes, or nasal drops, orspray, of said compound to the nasopharngeal airways, nasotracheal tube,endotracheal tube, or tracheostomy of said subject, such that atherapeutically effective amount of said compound contacts the airwaysof said subject either directly or via systemic absorption andcirculation.
 3. A method according to claim 1, wherein said compound isdelivered by administering an oral form of said compound, such that atherapeutically effective amount of said compound contacts the airwaysof said subject via systemic absorption and circulation.
 4. A methodaccording to claim 1, wherein said compound is delivered byadministering a nebulized aerosol or suspension of said compound to thenasopharyngeal airways, nasotracheal tube, endotracheal tube, ortracheostomy of said subject, such that a therapeutically effectiveamount of said compound contacts the airways of said subject eitherdirectly or via systemic absorption and circulation.
 5. A methodaccording to claim 1, wherein said compound is delivered byadministering a topical form of said compound to the airways via thenose, eyes, outer ear or nasopharyngeal airways of said subject, suchthat a therapeutically effective amount of said compound contacts theairways of said subject.
 6. A method according to claim 1, wherein saidcompound is delivered by administering an injected form of saidcompound, such that a therapeutically effective amount of said compoundcontacts the airways of said subject either directly or via systemicabsorption and circulation.
 7. A method according to claim 1, whereinsaid compound is delivered by administering a suppository form of saidcompound, such that a therapeutically effective amount of said compoundcontacts the airways of said subject via systemic absorption andcirculation.
 8. A method according to claim 1, wherein said compound isdelivered by administering an intra-operative instillation of a gel,cream, powder, foam, crystals or liquid suspension form of the activecompound such that a therapeutically effective amount of said compoundcontacts the airways either directly or via systemic absorption andcirculation.
 9. A method according to claim 1, wherein said compound isdelivered by administering a dry-powder aerosolized form of saidcompound, such that a therapeutically effective amount of said compoundcontacts the airways of said subject either directly or via systemicabsorption and circulation.
 10. A method according to claim 1, whereinsaid compound is administered in an amount sufficient to achieveconcentrations thereof on the surfaces of the airways of said subject toincrease the ciliary beat frequency of cilia on the surface of luminalepithelia cells, to increase the secretions of mucous by globet cells,to increase the chloride ion secretion to stimulate surfactant reductionand to promote the clearance of retained secretions.
 11. A methodaccording to claim 1, wherein said compound is administered in an amountsufficient to achieve concentrations on the surfaces of the airways ofsaid subject of from about 10-7 to about 10-1 moles/liter.
 12. A methodaccording to claim 1, wherein X₂ and X₃ are OH.
 13. A method accordingto claim 1, wherein R₁ is oxygen.
 14. A method according to claim 1,wherein R₂ is H.
 15. A method according to claim 1, wherein saidcompound of Formula I is selected from the group consisting of uridine5′-triphosphate, uridine 5′-O-(3-thiotriphosphate), 5-bromo-uridine 5′triphosphate and the pharmaceutically acceptable salts thereof.
 16. Amethod according to claim 1, wherein said compound of Formula II isselected from the group consisting of P1,P4-di(uridine-5′)tetraphosphate(U₂P₄) and P1,P4-di(adenosine-5′) tetraphosphate (A₂P₄) and substitutedderivatives and the pharmaceutically acceptable salts thereof.
 17. Amethod according to claim 1, wherein said compound of Formula III isselected from the group consisting of adenosine 5′-triphosphate,1,N6-ethenoadenosine 5′-triphosphate, adenosine 1-oxide 5′-triphosphateand the pharmaceutically acceptable salts thereof.
 18. A methodaccording to claim 1, wherein said compound of Formula IV is selectedfrom the group consisting of cytidine 5′-triphosphate (CTP),3,N4-ethenocytidine 5′-triphosphate and the pharmaceutically acceptablesalts thereof.
 19. A method of preventing or treating sinusitis in anasally-intubated patient, said method comprising: administering to thesubject a compound of Formula I, II, III or IV, or a pharmaceuticallyacceptable salt thereof, in a pharmaceutical carrier having an amount ofsaid compound effective to promote mucociliary clearance from thesinuses.
 20. A method of preventing or treating retained mucoussecretions in a bedridden or immobilized patient, said methodcomprising: administering to the subject a compound of Formula I, II,III or IV, or a pharmaceutically acceptable salt thereof, in apharmaceutical carrier having an amount of said compound effective topromote mucociliary clearance from the airways.
 21. A method accordingto claim 20, wherein the subject is placed in a lateral rotationtherapeutic bed which rotates the subject to further loosen mucoussecretions.